Over the past 30 years, medical attention has increasingly turned to the possibility of using naturally produced proteins as therapeutic drugs for the treatment of disease.
Recombinant DNA techniques have become the primary method for commercial production of many polypeptides and proteins because of the large quantities that can be produced in bacteria and other host cells. Recombinant protein production involves transfecting or transforming host cells with DNA encoding the desired exogenous protein and growing the cells under conditions favoring expression of the recombinant protein. E. coli and yeast are favored as hosts because they can be made to produce recombinant proteins at high titers (see, U.S. Pat. No. 5,756,672 (Builder et al.).
Numerous U.S. patents have been issued with respect to general bacterial expression of recombinant-DNA-encoded proteins (see, for example, U.S. Pat. Nos. 4,565,785; 4,673,641; 4,738,921; 4,795,706; 4,710,473). Unfortunately, the use of recombinant DNA techniques has not been universally successful. Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts are precipitated within the cells in dense aggregates, recognized as bright spots visible within the enclosure of the cells under a phase-contrast microscope. These aggregates of precipitated proteins are referred to as “refractile bodies,” and can constitute a significant portion of the total cell protein (Brems et al., Biochemistry (1985) 24: 7662.
Recovery of protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. For general review articles on retractile bodies, see Marston, supra; Mitraki and King, Bio/Technology, 7: 690 (1989); Marston and Hartley, Methods in Enzymol., 182: 264–276 (1990); Wetzel, “Protein Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian Amyloid,” in Stability of Protein Pharmaceuticals: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, Ahern and Manning (eds.) (Plenum Press, 1991); and Wetzel, “Enhanced Folding and Stabilization of Proteins by Suppression of Aggregation In Vitro and In Vivo,” in Protein Engineering—A Practical Approach, Rees, A. R. et al. (eds.) (IRL Press at Oxford University Press, Oxford, 1991). A need therefore exists for an alternative way of producing bioactive proteins.
One alternative to recombinant production of proteins involves the use of the principles of organic chemistry to synthesize proteins. Existing methods for the chemical synthesis of proteins include stepwise solid phase synthesis, and fragment condensation either in solution or on solid phase. The classic stepwise solid phase synthesis of Merrifield involves covalently linking an amino acid corresponding to the carboxy-terminal amino acid of the desired peptide chain to a solid support and extending the polypeptide chain toward the amino end by stepwise coupling of activated amino acid derivatives having activated carboxyl groups. After completion of the assembly of the fully protected solid phase bound peptide chain, the peptide-solid phase covalent attachment is cleaved by suitable chemistry and the protecting groups removed to give the product polypeptide.
There are unfortunately multiple disadvantages to the stepwise solid phase synthesis method, including the formation of solid-phase bound by products that result from incomplete reaction at the coupling and deprotection steps in each cycle. The longer the peptide chain, the more challenging it is to obtain high-purity well-defined products. The synthesis of proteins and large polypeptides by this route is a time-consuming and laborious task.
The solid phase fragment condensation approach (also known as segment condensation) was designed to overcome the difficulties in obtaining long polypeptides via the solid phase stepwise synthesis method. The segment condensation method involves preparation of several peptide segments by the solid phase stepwise method, followed by cleavage from the solid phase and purification of these maximally protected segments. The protected segments are condensed one-by-one to the first segment, which is bound to the solid phase. Often, however, technical difficulties are encountered in many of the steps of solid phase segment condensation. See E. Atherton, et al., “Solid Phase Fragment Condensation—The Problems,” in Innovation and Perspectives in Solid Phase Synthesis 11–25 (R. Epton, et al. 1990). For example, the use of protecting groups on segments to block undesired ligating reactions can frequently render the protected segments sparingly soluble, interfering in efficient activation of the carboxyl group. Limited solubility of protected segments also can interfere with purification of protected segments. See K. Akaji et al., Chem. Pharm. Bull.(Tokyo) 33:184–102 (1985). Protected segments are difficult to characterize with respect to purity, covalent structure, and are not amenable to high resolution analytical ESMS (electrospray mass spectrometry) (based on charge). Racemization of the C-terminal residue of each activated peptide segment is also a problem, except if ligating is performed at Glycine residues. Moreover, cleavage of the fully assembled, solid-phase bound polypeptide from the solid phase and removal of the protecting groups frequently can require harsh chemical procedures and long reaction times that result in degradation of the fully assembled polypeptide.
Segment condensation can be done in solution rather than on solid phase. See H. Muramatsu et al., Biochem. and Biophys. Res. Commn. 203(2):1131–1139 (1994). However, segment condensation in solution requires purification of segments prior to ligation as well as use of protecting groups on a range of different side chain functional groups to prevent multiple undesired side reactions. Moreover, the ligation in solution does not permit easy purification and wash steps afforded by solid phase ligations. Furthermore, the limitations with respect to solubility of protected peptide segments and protected peptide intermediate reaction products are exacerbated.
Chemical ligation of peptide segments has been explored in order to overcome the solubility problems frequently encountered with maximally protected peptide. Chemical ligation involves the formation of a selective covalent linkage between a first chemical component and a second chemical component. Unique, mutually reactive, functional groups present on the first and second components can be used to render the ligation reaction chemoselective. For example, the chemical ligation of peptides and polypeptides involves the chemoselective reaction of peptide or polypeptide segments bearing compatible Unique, mutually-reactive, C-terminal and N-terminal amino acid residues. Several different chemistries have been utilized for this purpose, examples of which include native chemical ligation (Dawson, et al., Science (1994) 266:776–779; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434), oxime forming chemical ligation (Rose, et al., J. Amer. Chem. Soc. (1994) 116:30–34), thioester forming ligation (Schnölzer, et al., Science (1992) 256:221–225), thioether forming ligation (Englebretsen, et al., Tet. Letts. (1995) 36(48):8871–8874), hydrazone forming ligation (Gaertner, et al., Bioconj. Chem. (1994) 5(4):333–338), and thiazolidine forming ligation and oxazolidine forming ligation (Zhang, et al., Proc. Natl. Acad. Sci. (1998) 95(16):9184–9189; Tam et al., WO 95/00846; U.S. Pat. No. 5,589,356) or by other methods (Yan, L. Z. and Dawson, P. E., “Synthesis of Peptides and Proteins without Cysteine Residues by Native Chemical Ligation Combined with Desulfurization,” J. Am. Chem. Soc. 2001; 123, 526–533, herein incorporated by reference; Gieselnan et al., Org. Lett. 2001 3(9):1331–1334; Saxon, E. et al., “Traceless” Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds. Org. Lett. 2000, 2, 2141–2143).
Of these methods, only the native chemical ligation approach yields a ligation product having a native amide (i.e. peptide) bond at the ligation site. The original native chemical ligation methodology (Dawson et al., supra; and WO 96/34878) has proven a robust methodology for generating a native amide bond at the ligation site. Native chemical ligation involves a chemoselective reaction between a first peptide or polypeptide segment having a C-terminal α-carboxythioester moiety and a second peptide or polypeptide having an N-terminal cysteine residue. A thiol exchange reaction yields an initial thioester-linked intermediate, which spontaneously rearranges to give a native amide bond at the ligation site while regenerating the cysteine side chain thiol. The primary drawback of the original native chemical ligation approach is that it requires an N-terminal cysteine, i.e., it only permits the joining of peptides and polypeptide segments possessing an N-terminal cysteine.
Notwithstanding this drawback, native chemical ligation of peptides with N-terminal amino acids other than cysteine has been reported (WO98/28434). In this approach, the ligation is performed using a first peptide or polypeptide segment having a C-terminal α-carboxythioester and a second peptide or polypeptide segment having an N-terminal N-{thiol-substituted auxiliary} group represented by the formula HS—CH2—CH2—O—NH-[peptide]. Following ligation, the N-{thiol substituted auxiliary} group is removed by cleaving the HS—CH2—CH2—O-auxiliary group to generate a native amide bond at the ligation site. One limitation of this method is that the use of a mercaptoethoxy auxiliary group can successfully lead to amide bond formation only at a glycine residue. This produces a ligation product that upon cleavage generates a glycine residue at the position of the N-substituted amino acid of the second peptide or polypeptide segment. As such, this embodiment of the method is only suitable if one desires the ligation product of the reaction to contain a glycine residue at this position, and in any event can be problematic with respect to ligation yields, stability of precursors, and the ability to remove the O-linked auxiliary group. Although other auxiliary groups may be used, for example the HSCH2CH2NH-[peptide], without limiting the reaction to ligation at a glycine residue, such auxiliary groups cannot be removed from the ligated product.
Accordingly, what is needed is a broadly applicable and robust chemical ligation system that extends native chemical ligation to a wide variety of different amino acid residues, peptides, polypeptides, polymers and other molecules by means of an effective, readily removable thiol-containing auxiliary group, and that joins such molecules together with a native amide bond at the ligation site. The present invention addresses these and other needs.